in vivo Protein Expression

The in vivo overexpression of protein using genetically engineered prokaryotic or eukaryotic cells remains the most efficient way to produce isotope-enriched protein and suitable for NMR analysis. Among prokaryotic expression systems, the use of the E. coli BL21(DE3) strain is by far the most popular and cost efficient. For some proteins, such as membrane proteins, overexpression in E. coli results in the recombinant protein being deposited into inclusion bodies in nonsoluble forms, which then requires refolding of the protein using detergents or lipids to gain a biochemically active form. To improve the probability of obtaining properly folded protein without the need for refolding, eukaryotic expression systems are used because these cell types contain more complex molecule machinery (e.g., chaperones) to aid in the folding proteins during expression. The most popular eukaryotic expression systems employ either yeast, insect, and mammalian cells. Uniform and most types of selective labeling are possible in all cell types.

Regardless of the method, proteins or complexes greater than ~25 kDa in size typically require deuterium enrichment in order to simplify spectra and reduce the deleterious effects of line-broadening associated with 1H dipolar coupling. Therefore, minimal media used to express such proteins in vivo must be formulated using deuterium oxide (D2O). For the investigations of large proteins or complexes, uniformly deuterated glucose as the carbon source is required.

Stable Isotopes for Biomolecular NMR

❛❛In our hands, CIL’s BioExpress 1000 worked like a charm. The cell growth rate and protein expression level essentially matched the results obtained with Luria broth, and the 15N labeling efficiency was excellent.❜❜

Tero Pihlajamaa, PhD | Institute of Biotechnology, University of Helsinki, Finland

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Celtone Complete

CIL’s Celtone is a top-selling fully rich media for the production of labeled recombinant proteins using E.coli. It is available as a powder for added stability (Celtone Base Powder) or as a liquid (Celtone Complete) for ease of use. Celtone Complete is a ready-to-use medium containing 10 g of Celtone powder per liter of media that does not require dilution or pH adjustment, and each lot is tested for sterility, cell growth, and protein expression.

Celtone Powder

CIL’s Celtone Powder is a labeled nutrient-rich additive for use with minimal media. The powdered media can be used at concentrations of 1 g to 10 g per liter to improve protein expression yields. Celtone Plus contains over twice the nutrients as the Celtone Base powder and contains uniformly enriched amino acids and peptides to promote growth for BL21 cells. Truly exceptional performance has been achieved using 10 g of Celtone Plus per liter of media, however, gains in yield have been apparent at concentrations as low as 1 g/L.

Frequently Asked Questions

How do I know which minimal media reagents to use to make a protein of a specific uniform labeling pattern? 


What algal strain is used to create BioExpress® 1000? Agmenelum quadriplicatum.

What algal strain is used to create Celtone® Powder and Celtone® Complete media? Chlorella vulgaris.

What is the difference between BioExpress and Celtone media? Both media are similar but as they are derived from two different algal strains, they have slight differences. To know which works better for a particular protein of interest, it is recommended to perform a small test experiment first.

What algal strain is used to create the Amino Acid Mixes product? Agmenelum quadriplicatum.

What algal strain is the Whole Algal Cells product? Agmenelum quadriplicatum.

Example References

Otten, R.; Chu, B.; Krewulak, K.D.; et al. 2010. Comprehensive and cost-effective NMR spectroscopy of methyl groups in large proteins. J Am Chem Soc, 132(9), 2952-2960. PMID: 20148553 
Studier, F. W. 2005. Protein production by auto-induction in high-density shaking cultures. Protein Expr Purif, 41(1), 207-234. PMID: 15915565
Tyler, R.C.; Sreenath, H.K.; Singh, S.; et al. 2005. Auto-induction medium for the production of [U-15N]- and [U-13C, U-15N]-labeled proteins for NMR screening and structure determination. Protein Expr Purif, 40(2), 268-278. PMID: 15766868